Abhijit Mazumder

Effects of Uracil Incorporation, DNA Mismatches, and Abasic Sites on Cleavage and Religation Activities of Mammalian Topoisomerase I

Abasic sites and deamination of cytosine to uracil are probably the most common types of endogenous DNA damage. The effects of such lesions on DNA topoisomerase I (top1) activity were examined in oligonucleotides containing a unique top1 cleavage site. The presence of uracils and abasic sites within the first 4 bases immediately 5′ to the cleavage site suppressed normal top1 cleavage and induced new top1 cleavage sites. Uracils immediately 3′ to the cleavage site increased cleavage and produced a camptothecin mimicking effect. A mismatch with a bulge or abasic sites immediately 3′ to the top1 cleavage site irreversibly trapped top1 cleavable complexes in the absence of camptothecin and produced a suicide cleavage complex. These results demonstrate that top1 activity is sensitive to physiological, environmental, and pharmacological DNA modifications and that top1 can act as a specific mismatch- and abasic site-nicking enzyme.

Trapping of Mammalian Topoisomerase I and Recombinations Induced by Damaged DNA Containing Nicks or Gaps IMPORTANCE OF DNA END PHOSPHORYLATION AND CAMPTOTHECIN EFFECTS

We used purified mammalian topoisomerases I (top1) and oligonucleotides containing a unique top1 cleavage site to study top1-mediated cleavage and recombination in the presence of nicks and short gaps mimicking DNA damage. In general, top1 cleavage was not induced opposite to the nicks, and nicks upstream from the top1 cleavage site suppressed top1 activity. Irreversible top1 cleavage complexes (“suicide products” or “aborted complexes”) were produced in DNA containing nicks or short gaps just opposite to the normal top1 cleavage site. Camptothecin enhanced the formation of the aborted top1 complexes only for nicks downstream from the cleavage site. These aborted (suicide) complexes can mediate DNA recombination and promote illegitimate recombination by catalyzing the ligation of nonhomologous DNA fragments (acceptors). We report for the first time that top1-mediated recombination is greatly enhanced by the presence of a phosphate at the 5′ terminus of the top1 aborted complex (donor DNA). By contrast, phosphorylation of the 3′ terminus of the gap did not affect recombination. At concentrations that strongly enhanced inhibition of intramolecular religation, resulting in an increase of top1 cleavable complexes, camptothecin did not reduce recombination (intermolecular religation). Nicks or gaps with 5′-phosphate termini would be expected to be produced directly by ionizing radiations or by processing of abasic sites and DNA lesions induced by carcinogens or drugs used in cancer chemotherapy. Thus, these results further demonstrate that DNA damage can efficiently trap top1-cleavable complexes and enhance top1-mediated DNA recombination.

HIV-1 Integrase as a Target for Antiviral Drugs:

Retrovirus integration requires at least two viral components, one of the three retroviral enzymes, integrase, and cis-acting sequences at the ends of the retroviral DNA termini U3 and U5 ends of the long terminal repeats. Because the virus cannot replicate without integration into a host chromosome, integrase is a logical therapeutic target. Therapeutic inhibitors already exist for reverse transcriptase and protease, and attacking the virus on these sites together has already proven effective for combination therapy. Thus, the discovery of integrase inhibitors should provide an additional benefit. Screening and pharmacology of anti-integrase drugs is facilitated by the cloning and expression of recombinant retroviral integrases and their use in a series of in vitro assays that mimic integration in vivo. This review first describes the integration reactions in the retrovirus life cycle and the integrase protein. Then we provide a comprehensive review of the inhibitors identified to date.

Chemical Trapping of Ternary Complexes of Human Immunodeficiency Virus Type 1 Integrase, Divalent Metal, and DNA Substrates Containing an Abasic Site IMPLICATIONS FOR THE ROLE OF LYSINE 136 IN DNA BINDING

We report a novel assay for monitoring the DNA binding of human immunodeficiency virus type 1 (HIV-1) integrase and the effect of cofactors and inhibitors. The assay uses depurinated oligonucleotides that can form a Schiff base between the aldehydic abasic site and a nearby enzyme lysine ε-amino group which can subsequently be trapped by reduction with sodium borohydride. Chemically depurinated duplex substrates representing the U5 end of the HIV-1 DNA were initially used. We next substituted an enzymatically generated abasic site for each of 10 nucleotides normally present in a 21-mer duplex oligonucleotide representing the U5 end of the HIV-1 DNA. Using HIV-1, HIV-2, or simian immunodeficiency virus integrases, the amount of covalent enzyme-DNA complex trapped decreased as the abasic site was moved away from the conserved CA dinucleotide. The enzyme-DNA complexes formed in the presence of manganese were not reversed by subsequent addition of EDTA, indicating that the divalent metal required for integrase catalysis is tightly bound in a ternary enzyme-metal-DNA complex. Both the N- and C-terminal domains of integrase contributed to efficient DNA binding, and mutation of Lys-136 significantly reduced Schiff base formation, implicating this residue in viral DNA binding.

2-Mercaptobenzenesulphonamides as novel inhibitors of human immunodeficiency virus type 1 integrase and replication

An obligatory requirement in the retroviral life cycle is the integration of the viral dsDNA into the host chromosome, a process performed by viral integrase. The retroviral integrase is able to catalyse at least three discrete enzymatic steps. Two of these steps, 3′ processing and DNA strand transfer, can be measured in an in vitro assay in the presence of a duplex oligonucleotide corresponding to the viral long terminal repeat, recombinant integrase and the divalent cations, Mg2+ or Mn2+. This assay provides an efficient means of testing integrase inhibitors. As part of our continuous effort in developing novel inhibitors we examined a series of 2-mercaptobenzenesulphonamides (MBSAs) for their inhibitory activity against human immunodeficiency virus type 1 (HIV-1) integrase. From the list of compounds tested in an assay specific for HIV-1 integrase, 26 compounds inhibited the 3′ processing and strand transfer step with 50% inhibitory concentration (IC50) values below 25 μM. All the thioether derivatives were inactive. These results were further compared with the ability of MBSAs to protect HIV-1-infected T4 lymphocyte CEM cells. Among 68 compounds tested, 27 exhibited antiviral activity in cell-based assays with therapeutic indices of 1-16. All the MBSAs with antiviral activity were also effective inhibitors of recombinant HIV-1 integrase. Several aromatic disulphides were also tested and found to exhibit moderate antiviral and anti-integrase activities. These data demonstrate that MBSAs can be developed as inhibitors of HIV-1 integrase with the potential for antiviral activity.